通用真菌引物和探针检测人全血标本中侵袭性真菌DNA载量的方法

Detection of invasive fungi DNA load in human whole blood samples with universal fungi primers and probe

目的建立实时定量PCR（RQ-PCR）以真菌通用引物和探针快速准确检测人全血标本中侵袭性真菌DNA载量的方法，并与细菌相鉴别及进行初步临床应用。方法选择临床常见的真菌基因组多拷贝基因5.8S rDNA作为靶基因设计特异性通用真菌引物和TaqMan探针，采用QIAamp血液DNA小提试剂盒提取多种致病真菌基因组DNA，建立20 μl RQ-PCR反应体系，对含有不同载量致病真菌的模拟人全血标本和71份外科发热患者全血标本进行侵袭性真菌基因组的定量检测。结果本方法的特异性良好，检测限为101拷贝/μl上机待测液 （即约105 拷贝/ml 全血）；检测灵敏度和特异度分别为95.5%和97.6%，阳性预告值和阴性预告值分别为98.7%和92.0%；标准曲线R2在0.9931~0.9977；批内及批间平均变异系数分别为（10.4±4.0）%和（27.9±2.0）%；人血标本中真菌基因组DNA平均回收率为（91.0±7.6）%，相对回收率平均变异系数为（14.9±4.0）%。71份外科发热患者血标本中未检测出侵袭性真菌基因组。结论RQ-PCR可以借通用真菌引物和TaqMan探针快速、特异、灵敏地定量检测人血标本中侵袭性真菌DNA的载量并可与细菌相鉴别，且有着较好的准确度与精密度。外科发热患者血中侵袭性真菌基因组的存在率可能很低。

ObjectiveTo establish a real-time quantitative PCR （RQ-PCR） assay for fast detection of invasive fungi DNA in human whole blood samples with universal fungi primers and probe. MethodsThe universal fungi primers and the TaqMan-probe were designed on the basis of the multi-copy 5.8S region of the rDNA of the clinically most common invasive fungi. The invasive fungi genomic DNA were extracted with QIAamp DNA Blood Mini Kit. A 20 μl RQ-PCR amplification system was established, and the simulated blood samples containing various given load of invasive fungi genome and the 71 whole blood samples of the surgical febrile patients were examined. ResultsThe detection limit is 101 copies/μl amplification mixture, namely 105 copies /ml whole blood. The sensitivity and the specificity were 95.5% and 97.6%, respectively; and the positive predictive value and negative predictive value were 98.7% and 92.0%, respectively. The correlation coefficient of standard curve was between 0.9931 and 0.9977. The intra- and the inter-assay average coefficients of variation were （10.4±4.0） % and （27.9±2.0） %, respectively. The average relative recovery rate of fungi genomic DNA in blood samples was （91.0±7.6）%, and the average coefficients of variation of the relative recovery rate was （14.9±4.0） %. No fungi DNA was detected among the 71 blood samples of the surgical febrile patients. ConclusionsThe RQ-PCR assay for fast quantitative detection of invasive fungal DNA in human whole blood samples with the universal fungi primers and the TaqMan-probe was of high sensitivity, specificity, accuracy and precision, and is able to discriminate fungi from bacteria. The invasive fungi genome was not detected in this group of surgical patients, which may imply the less possibility of fungi translocation in the surgical febrile patients.